VEGF is a naturally occurring compound that is produced in follicular or folliculo-stellate cells (FC), a morphologically well characterized population of granular cells. The FC are stellate cells that send cytoplasmic processes between secretory cells.
Several years ago a heparin-binding endothelial cell-growth factor called vascular endothelial growth factor (VEGF) was identified and purified from media conditioned by bovine pituitary follicular or folliculo-stellate cells. See Ferrara et al., Biophys. Res. Comm. 161, 851 (1989).
Although a vascular endothelial cell growth factor could be isolated and purified from natural sources for subsequent therapeutic use, the relatively low concentrations of the protein in FC and the high cost, both in terms of effort and expense, of recovering VEGF proved commercially unavailing. Accordingly, further efforts were undertaken to clone and express VEGF via recombinant DNA techniques. The embodiments of that research are set forth in the patent applications referred to supra; this research was also reported in the scientific literature in Laboratory Investigation 72, 615 (1995), and the references cited therein.
In those applications there is described an isolated nucleic acid sequence comprising a sequence that encodes a vascular endothelial cell growth factor having a molecular weight of about 45,000 daltons under non-reducing conditions and about 23,000 under reducing conditions as measured by SDS-PAGE. Both the DNA and amino acid sequences are set forth in figures forming a part of the present application—see infra.
VEGF prepared as described in the patent applications cited supra, is useful for treating conditions in which a selected action on the vascular endothelial cells, in the absence of excessive tissue growth, is important, for example, diabetic ulcers and vascular injuries resulting from trauma such as subcutaneous wounds. Being a vascular (artery and venus) endothelial cell growth factor, VEGF restores cells that are damaged, a process referred to as vasculogenesis, and stimulates the formulation of new vessels, a process referred to as angiogenesis.
VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189 and 206 amino acids per monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The heparin-binding forms of VEGF can be cleaved in the carboxy terminus by plasmin to release (a) diffusible form(s) of VEGF. Amino acid sequencing of the carboxy terminal peptide identified after plasmin cleavage is Arg110–Ala111. Amino terminal “core” protein, VEGF (1-110) isolated as a homodimer, binds neutralizing monoclonal antibodies (4.6.1 and 2E3) and soluble forms of FLT-1, KDR and FLK receptors with similar affinity compared to the intact VEGF165 homodimer. VEGF contains a C-terminal heparin binding domain that generally spans the C-terminus beginning beyond about amino acid 120. Generally this domain carries a relatively large number of positively charged amino acids.
The present invention is predicated upon initial research results that compared the heparin binding properties of VEGF derived respectively from recombinant Chinese hamster ovary (CHO) and E. coli cells. This research resulted in the finding that the CHO-derived VEGF material contained various C-terminal “processing” resulting in forms having different lengths with respect to the heparin binding C-terminal domain versus the substantially full-length material derived via E. coli production. Such C-terminal processing includes internal clips, i.e., cleaved sites, within the heparin binding domain that may alter the secondary and tertiary structure of the heparin binding domain so as to decease its affinity for heparin and endogenous heparan sulfate proteoglycans.
Further research indicated that such C-terminal processing of VEGF resulted in variants exhibiting slower rates of clearance and smaller volumes of distribution. Although these processed variants still possess at least a portion of the heparin binding domain, the modified domain bound heparin and heparin sulfate proteoglycans with lower affinity. The resultant effect was that less VEGF was cleared by non-specific target organs such as the liver.
It was therefore a further object of this research to produce VEGF variants that would have C-terminal variations with consequential varying heparin binding affinity resulting in variants of VEGF having a reduced clearance rate and hence longer retention within the body after systemic administration such that lower doses of the material were available for systemic administration for therapeutic effect. The intellectual property of such research is the subject of the present invention.